Published online before print February 26, 2004, doi: 10.1161/01.RES.0000124300.76171.C9
© 2004 American Heart Association, Inc.
Molecular Medicine |
Notch Activation Results in Phenotypic and Functional Changes Consistent With Endothelial-to-Mesenchymal Transformation
From the Department of Pathology and Laboratory Medicine (M.N., I.P., A.K.) and Experimental Medicine Program (G.M., K.N., L.C., A.K.), University of British Columbia; Department of Medical Biophysics (M.N., G.M., K.N., L.C., I.P., R.E.D., A.K.), Terry Fox Research Laboratories (R.M., P.A.H.), and Department of Pathology and Laboratory Medicine (A.K.), British Columbia Cancer Agency; and Department of Pathology (R.S., K.D.-Z.), Division of Neuropathology, University of British Columbia and Vancouver Hospital and Health Sciences Centre, Vancouver, British Columbia, Canada; Stem Cell Research Laboratory (L.L.), Stowers Institute for Medical Research, Kansas City, Mo; and Department of Bioengineering (B.B.), University of Washington, Seattle, Wash.
Correspondence to Aly Karsan, Department of Pathology and Laboratory Medicine and Experimental Medicine Program, University of British Columbia, Vancouver, BC V6T 2B5, Canada. E-mail akarsan{at}bccrc.ca
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Various studies have identified a critical role for Notch signaling in cardiovascular development. In this and other systems, Notch receptors and ligands are expressed in regions that undergo epithelial-to-mesenchymal transformation. However, there is no direct evidence that Notch activation can induce mesenchymal transdifferentiation. In this study we show that Notch activation in endothelial cells results in morphological, phenotypic, and functional changes consistent with mesenchymal transformation. These changes include downregulation of endothelial markers (vascular endothelial [VE]-cadherin, Tie1, Tie2, platelet-endothelial cell adhesion molecule-1, and endothelial NO synthase), upregulation of mesenchymal markers (
-smooth muscle actin, fibronectin, and platelet-derived growth factor receptors), and migration toward platelet-derived growth factor-BB. Notch-induced endothelial-to-mesenchymal transformation does not seem to require external regulation and is restricted to cells expressing activated Notch. Jagged1 stimulation of endothelial cells induces a similar mesenchymal transformation, and Jagged1, Notch1, and Notch4 are expressed in the ventricular outflow tract during stages of endocardial cushion formation. This is the first evidence that Jagged1-Notch interactions induce endothelial-to-mesenchymal transformation, and our findings suggest that Notch signaling may be required for proper endocardial cushion differentiation and/or vascular smooth muscle cell development.
Key Words: endothelial-to-mesenchymal transformation • Notch • Jagged1 • endocardial cushion
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The Notch signaling pathway plays a critical role during development. Four mammalian Notch receptors (Notch1 through 4) and 5 Notch ligands (Delta-like [Dll]-1, Dll3, Dll4, Jagged1, and Jagged2) have been identified. Notch receptor-ligand interaction results in a series of proteolytic cleavages of the Notch receptor, producing a C-terminal intracellular fragment (NotchIC) that translocates to the nucleus. In the nucleus, NotchIC binds to the transcriptional repressor CBF1/RBP-J
, thereby derepressing or coactivating the expression of various lineage-specific genes.1 Perturbation of the Notch pathway has been implicated in the pathogenesis of various cardiovascular diseases in humans.2 Of interest, patients with Jagged1 mutations (Alagille syndrome) display congenital cardiovascular anomalies that seem to be secondary to faulty endocardial cushion formation.3–6 In the mouse, Notch1-deficient embryos demonstrate severe vascular developmental defects, which are exacerbated in Notch1/Notch4 double-mutant embryos.7 Constitutive activation of Notch4 also causes defects in vascular remodeling.8,9 Mice that are rendered null for Jagged1 die from hemorrhage early during embryogenesis, whereas mice that are doubly heterozygous for a Jagged1-null allele and a Notch2 hypomorphic allele exhibit cardiac anomalies similar to those seen in Alagille syndrome.10,11 Genes that lie downstream of Notch activation, such as the basic helix-loop-helix factor, HRT2/HEY2, have also been implicated in cardiovascular development.12,13
Notch receptors and their ligands have been localized to the vasculature.14 Notch receptors have also been observed in the endocardium, and the Notch ligand Jagged1 is present on endocardial and periendocardial cells of the cardiac cushions.15,16 The endocardial cushion is a specialized embryonic tissue that gives rise to the cardiac valves and membranous septa. A critical event in cardiac cushion formation is a differentiation process referred to as endothelial-to-mesenchymal transformation (EMT), which is a specific form of epithelial-to-mesenchymal transformation.17,18 In the cardiovascular system of the adult, mesenchymal cells derived from the transformation of a subset of cardiac valve endothelial cells may also be necessary for maintenance of the leaflet architecture.19 Furthermore, EMT may play a role in the development of neointimal lesions in transplant atherosclerosis and restenosis.20
Intercellular signaling between Notch receptors and ligands is critical for cell fate determination by influencing cell proliferation, differentiation, and apoptosis.21 Notch members and their ligands are expressed in various regions that undergo EMT in order for development to proceed appropriately.22,23 Our studies demonstrate that Notch activation in endothelial cells promotes mesenchymal transformation and suggest that Jagged1-Notch interactions may participate in endocardial cushion formation by inducing EMT.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cell Culture and Reagents
The HMEC-1 microvascular endothelial cell line, hereafter referred to as HMEC, was provided by the Centers for Disease Control and Prevention (Atlanta, Ga) and cultured as previously described.9 Human umbilical vein endothelial cells (HUVECs) were isolated and cultured as previously described.24 Human aortic endothelial cells (HAECs) were purchased and cultured in supplemented endothelial growth media (Clonetics). Ovine endocardial cells (OECs) were isolated from sheep cardiac ventricles by treatment with collagenase (45 minutes at 37°C). DiI-acetylated LDL uptake and expression of endothelial markers (vascular endothelial [VE]-cadherin and platelet-endothelial cell adhesion molecule [PECAM-1]) were confirmed. OECs were maintained in Waymouth’s media (Gibco) with 10% FBS and antibiotics.
Gene Transfer
Endothelial cells were transduced using the retroviral vector MSCV-IRES-YFP (MIY) (gift from R.K. Humphries, British Columbia Cancer Agency, Vancouver, BC). cDNA constructs encoding the C-terminal HA-tagged Notch4 intracellular domain, Notch1 intracellular domain (gift of S. Artavanis-Tsakonas, Harvard Medical School, Charlestown, Mass), and full-length Jagged1 were cloned into MIY. Endothelial cells were transduced as previously described.25
Transmission Electron Microscopy
Endothelial cultures were processed as previously described.26 Briefly, cultures were washed with M199, fixed in 2.5% glutaraldehyde/2% paraformaldehyde in 0.2 mol/L sodium cacodylate buffer for 1 hour, post-fixed in 1% OsO4 for 1 hour, stained en bloc with uranyl magnesium acetate, dehydrated, and embedded in Epon-Araldite. Blocks cut from the embedded cultures were reembedded for cross-sectioning. Thin sections were stained with uranyl acetate and lead citrate and viewed on a Zeiss EM 10 microscope.
Immunoblotting
Cells were lysed, and 50 µg of total protein was analyzed by SDS-PAGE. The monoclonal antibody against the HA epitope was purchased from BAbCo, and anti–VE-cadherin, anti–PECAM-1, anti-Tie1, and anti-Tie2 antibodies were all from Santa Cruz Biotechnology. Anti-endothelial NO synthase (eNOS/NOS type III) and anti-fibronectin antibodies were purchased from Transduction Laboratories, and anti–-smooth muscle actin (SMA) antibody was obtained from Cymbus Biotechnology (Hampshire, UK). Anti-phospho-Smad2 and anti-total-Smad2 antibodies (gift from N. Khalil, University of British Columbia, Vancouver, BC) were manufactured by UBI.
Migration Assay
The ability of endothelial cells to migrate toward platelet-derived growth factor (PDGF)-BB (20 ng/mL) was measured by a modified Boyden chamber assay as previously described.9
Immunocytochemistry
Cells were fixed in 4% paraformaldehyde and blocked/permeablized in 4% FCS/0.2% Triton X-100/PBS. Secondary antibodies were Alexa 594-conjugated. To quantitate the proportion of SMA-positive cells, at least five high-power fields (comprising at least 200 cells) were evaluated, and the proportion of SMA-positive cells expressed were as a percentage of the total number of DAPI-stained nuclei in each field.
Flow Cytometry
Cells were trypsinized, washed in PBS, and fixed in 4% paraformaldehyde. Cells were blocked/permeabilized, stained with anti-SMA antibody, and analyzed on an EPICS ELITE-ESP flow cytometer (Beckman Coulter).
Luciferase Assays
Endothelial cells were transfected by electroporation (1.5x106 cells) with 1 to 5 µg of plasmid DNA as previously described.27 Forty-eight hours after transfection, dual-luciferase reporter assays were performed according to manufacturer’s recommendations (Promega Corporation). The CBF1-dependent reporter, 4xCBF1wt-LUC, was a gift from S.D. Hayward (Johns Hopkins School of Medicine, Baltimore, Md).28 The SMA promoter-reporter construct encompassing a 5.4-kb region comprising –2555 to +2813 of the rat SMA gene was a gift from F. Dandre and G.K. Owens (University of Virginia, Charlottesville, Va). Transfections were normalized by transfecting cells with 50 ng of the Renilla luciferase plasmid pRL-CMV (Promega).
In Situ Hybridization
The murine Jagged1 probe was a gift from S.E. Egan (Hospital for Sick Children, Toronto, Ontario), and the Notch1 and Notch4 probes were a gift from J. Rossant (Samuel Lunenfeld Research Institute, Toronto, Ontario). For whole-mount in situ hybridization, embryonic hearts were fixed overnight at 4°C in 4% paraformaldehyde in PBS, dehydrated in methanol, and stored at –20°C. For hybridizations, embryonic hearts were processed as described.29
Reverse Transcriptase–Polymerase Chain Reaction
Total RNA was isolated using TRIzol Reagent (Invitrogen), DNase-treated, and reverse-transcribed to cDNA, followed by polymerase chain reaction (PCR) (see the online data supplement for primers and annealing conditions, available at http://circres.ahajournals.org). A control reaction, omitting reverse transcriptase (RT), was performed for each RNA sample to verify the absence of genomic DNA.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Activated Notch Induces Endothelial-to-Mesenchymal Transformation
During the course of a previous study, we noted that HMECs expressing Notch4IC (HMEC-Notch4IC) lost the characteristic cobblestone morphology of confluent endothelial cells.9 As seen in Figure 1A, HMEC-Notch4IC formed multilayered cultures suggesting loss of endothelial phenotype and potential transformation to mesenchymal cells. Transmission electron microscopy confirmed that HMEC-Notch4IC failed to form monolayers and proper cell-to-cell junctions and showed marked overlapping with infrequent, rudimentary cell contacts (Figures 1B, panel i, and 1B, panel ii). In contrast, HMEC-vector cells retained their capacity to form junctions (Figure 1B, panel iii).
|
VE-cadherin is a cell adhesion molecule that is localized to the interendothelial region and is required for the formation of adherens junctions.30 Immunoblotting for VE-cadherin demonstrated significant downregulation of this critical junctional molecule, suggesting mesenchymal transformation (Figure 1C). Furthermore, we noted a reduction in expression, to varying degrees, of several other endothelial-specific proteins (PECAM-1, Tie1, Tie2, and endothelial NOS) (Figure 1C). In addition to the loss of endothelial phenotype, EMT implies the acquisition of mesenchymal markers.19,31–35 To determine whether Notch4-activated cells upregulate mesenchymal proteins, we examined expression of SMA, fibronectin, and PDGF receptors in HMEC-Notch4IC. Immunoblotting shows induction of all three proteins in HMEC-Notch4IC (Figure 1C).
PDGF is a known chemotactic factor for mesenchymal cells, and in particular, PDGF-BB plays a role in recruitment of mesenchymal cells during vascular development.35,36 Hence, we examined the chemotactic response of HMEC-Notch4IC or HMEC-vector to PDGF-BB. HMEC-Notch4IC was able to migrate toward PDGF-BB in a modified Boyden chamber assay, whereas vector-transduced cells did not (Figure 1D). Thus, Notch4 activation in endothelial cells induces morphological, phenotypic, and functional changes observed during EMT.
Several studies have shown that aortic endothelium can differentiate into mesenchymal-like cells in vitro.31,37 More recent work has demonstrated that HUVECs also retain the potential to differentiate into mesenchymal cells.19,38 In addition to Notch4, Notch1 is also expressed in endothelial cells.14 Hence, we transduced HMECs, HAECs, and HUVECs with Notch4IC, Notch1IC, or empty vector. Our data demonstrated that activated Notch1, as well as Notch4, had the potential to induce EMT in endothelial cells from different vascular beds, as determined by morphological, immunophenotypic, and functional criteria (online Figure 1 and data not shown). Because of the critical requirement for EMT during cardiac cushion formation, we determined whether activated Notch was also able to induce EMT in cardiac endothelial cells. OECs were transduced with the empty vector, Notch4IC, or Notch1IC. OECs also underwent a morphological transformation, as witnessed by loss of uniform cell shape, loss of intercellular contacts, cellular polarization, formation of filopodia (Figure 2A), and the induction of SMA (Figure 2B). Downregulation of endocardial proteins such as PECAM-1 was also confirmed (data not shown).
|
To determine the efficiency of Notch-induced EMT in different endothelial types, we quantitated SMA-positive cells by immunofluorescent staining 10 to 14 days after transduction. HAECs and OECs demonstrated the greatest capacity to undergo Notch-induced EMT (Figure 2C). Interestingly, in all endothelial types, Notch1 showed greater efficacy in inducing EMT. Taken together, these results indicate that activated Notch is able to induce mesenchymal transformation in endothelial cells from various vascular beds and suggest that different Notch members may play similar functional roles in EMT.
Notch-Induced EMT Is Restricted to Cells Expressing Activated Notch
Activated Notch may induce EMT in conjunction with activation of other signaling molecules. In this regard, transforming growth factor-ß (TGF-ß) has been suggested to play an important role in EMT in the endocardial cushion, as well as in ovine and human valvular and bovine aortic endothelial cells.19,31,39 Primary human endothelial cells transduced with Notch4IC or Notch1IC showed variable levels of TGF-ß expression with no consistent increase induced by Notch activation, although HMEC-Notch4IC did show increased TGF-ß2 secretion (data not shown). To determine whether TGF-ß stimulation was sufficient to induce EMT, we treated HMECs and HAECs with recombinant TGF-ß1 or TGF-ß2 (5 ng/mL) for up to 28 days. Consistent with studies performed on human vascular endothelial cells, treatment of HMECs and HAECs with exogenous TGF-ß1 or TGF-ß2 did not induce morphological changes of EMT or expression of SMA (data not shown).19 Furthermore, inhibition of TGF-ß activity with a pan-anti–TGF-ß–neutralizing antibody did not inhibit or reduce the Notch4IC- or Notch1IC-induced morphological changes or SMA expression in either HMECs or HAECs despite the ability of this antibody to inhibit phosphorylation of Smad2 (Figure 3).
|
To determine whether NotchIC-transduced endothelial cells secrete other soluble factors that are capable of inducing phenotypic changes, we added 3-day conditioned medium from HMEC-vector and HMEC-Notch4IC to parental HMECs or primary HAECs. We did not observe morphological changes or SMA expression in HMECs or HAECs treated daily with conditioned medium from Notch4IC-transduced HMECs over a 28-day period (data not shown). The above findings suggest that Notch-induced mesenchymal transformation does not depend on paracrine factors and is likely restricted to cells expressing activated Notch.
To confirm that Notch-induced mesenchymal transformation occurs only in cells expressing activated Notch, HMECs were infected with a retroviral vector (MIY) that contains yellow fluorescence protein (YFP) linked to the transgene through an internal ribosomal entry site. Only the YFP-positive (Notch4IC-expressing) cells expressed SMA, as determined by flow cytometry (Figure 4A). This finding was confirmed by high-purity cell sorting of YFP-positive and YFP-negative subpopulations of both Notch4IC- and vector-transduced cells. Immunoblotting of the sorted populations showed downregulation of endothelial-specific proteins and upregulation of mesenchymal markers only in YFP-positive HMEC-Notch4IC (Figure 4B). Studies using Notch1IC-expressing HUVECs similarly demonstrated that only YFP-positive (Notch1IC-expressing) HUVECs expressed SMA (online Figure 2). Thus, only endothelial cells that express activated Notch undergo EMT, and external factors do not seem to be required for this process in vitro. However, additional studies assessing the role of TGF-ß family members will be required to understand the interaction of this pathway with Notch signaling during EMT.
|
Jagged1 Induces EMT
The Notch ligand, Jagged1, is expressed on endothelial and smooth muscle cells during development and in the adult.14 Jagged1 mutations are associated with congenital cardiac anomalies that may be secondary to defective endocardial cushion development.3–6 To determine whether Jagged1-Notch interactions can induce EMT, we expressed wild-type Jagged1 in HMECs (HMEC-Jagged1). HMEC-Jagged1 or HMEC-vector were cocultured with parental HMECs, and expression of endothelial markers was analyzed by immunoblotting. In cocultures with HMEC-Jagged1, VE-cadherin, PECAM-1 and Tie2 were downregulated and fibronectin was upregulated (Figure 5A). We verified that Jagged1-Notch interactions induced SMA expression by immunofluorescent staining (Figure 5B). To confirm activation of the Notch pathway by Jagged1, HMEC-Jagged1 cocultures and HMEC-vector cocultures were transiently transfected with a CBF1-dependent promoter-luciferase construct. HMEC-Jagged1 cocultures demonstrated 4.5-fold higher CBF1-luciferase activity compared with HMEC-vector cocultures, demonstrating that enforced expression of Jagged1 activates CBF1-dependent Notch signaling (Figure 5C). The presence of a conserved consensus CBF-1 binding site that is located 64 to 59 nucleotides upstream of the cap site in the SMA promoter suggests that activation of Notch may directly induce SMA transcription. Promoter-luciferase assays using Notch4IC- or Notch1IC-transduced HMECs demonstrated a Notch-dependent increase in SMA promoter activity that was confirmed by induction of SMA mRNA by RT-PCR (data not shown). Examination of HMEC-Jagged1 cocultures also demonstrated a 2.5-fold increase in SMA promoter-luciferase activity over HMEC vector, indicating that Jagged1-Notch interactions are capable of upregulating SMA expression (Figure 5D). These results do not prove a direct transcriptional regulation of SMA by putative Notch-CBF1 complexes but suggest that increased expression of SMA may be attributable to induction of transcription. Taken together, our results support the hypothesis that Jagged1-Notch interactions contribute to the transdifferentiation of endothelial cells to a mesenchymal phenotype.
|
Notch1, Notch4, and Jagged1 Are Expressed in the Early Developing Heart
Because our data demonstrate that in vitro Jagged1-Notch interactions induce EMT, we sought to determine whether Notch1, Notch4, and Jagged1 were expressed in areas where EMT is occurring in the developing heart. EMT begins at approximately embryonic day (E) 9.5 in the atrioventricular endocardial cushions and at E10.5 in the ventricular outflow tract.40 At E10.5, Notch1 is expressed throughout the ventricles in the forming trabeculae (Figure 6H). Low-level expression is also observed in the inner endocardial layer of the atria (Figure 6C and data not shown). The highest level of Notch1 expression is present in the endothelium of the outflow tract at the time when EMT is commencing in this region (Figures 6C, 6D, and 6H). Notch4 exhibits low levels of expression throughout the heart at E10.5 (Figure 6F) and is selectively observed in regions of presumptive valve leaflet development in the outflow tract at E11.5 (Figure 6G).
|
Jagged1 expression can be detected as early as E9.5 in the bulbus cordis of the developing heart (data not shown), the region of the heart that will form the right ventricle and the outflow tract. By E10.5, Jagged1 expression can be detected throughout the heart, including the atria and ventricles, with significantly higher levels observed in the outflow tract (Figure 6J). In the outflow tract, expression is observed in both the endothelial cells and in the surrounding mesenchyme, with notably higher expression in the anterior portion of the outflow tract (Figure 6K). A clear boundary between high expression and low expression is seen at the region of the outflow tract where separation of the pulmonary artery and the aorta begins. Expression of Jagged1 in the outflow tract is maintained at E11.5 (Figure 6L). To examine expression of Notch receptors and ligands earlier in cardiac development, RT-PCR was performed on mouse hearts harvested at E9.5 and separated into atria, ventricles, and bulbus cordis (outflow tract). As seen in Figure 6M, Jagged1 and all four Notch receptors are strongly expressed in the bulbus cordis.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our findings demonstrate that expression of activated Notch1 or Notch4 in endothelial cells causes transdifferentiation to a mesenchymal phenotype and implicate Jagged1-Notch interactions in promoting EMT. One role for Notch-mediated EMT may occur during endocardial cushion formation. Endocardial cushion development is a key process in the formation of the valves and membranous septum, which partitions the heart tube into an atrium and ventricle and the common ventricular outflow tract into dorsal aorta and pulmonary artery.17,18 Given that Notch receptors and the ligand Jagged1 are expressed in regions of the heart where EMT is required for proper development, the results described herein support a role for Jagged1-Notch interactions in promoting mesenchymal transformation during cardiac cushion formation and subsequent valvuloseptal development.15,16 Interestingly,
70% of patients affected by Alagille syndrome, a disorder with pleiotropic developmental abnormalities, have mutations in the Jagged1 gene.3–6 The most frequent cardiac anomaly in these patients is peripheral pulmonic stenosis. However, abnormalities that implicate defective endocardial cushion development are also described.3–6 In particular, a recent study reported that 13% of patients with Alagille syndrome are affected by tetralogy of Fallot.5 Furthermore, mutations of Jagged1 are associated with defects of cardiac cushion development independent of the other developmental anomalies reported by Alagille.6,41 We speculate that defective Notch activation and consequent attenuation of EMT may play a role in the pathogenesis of atrioventricular defects associated with Jagged1 mutations. There is accumulating evidence that endothelial cells can transdifferentiate into smooth muscle cells during developmental and pathological processes.42 Labeling studies in quail embryos showed that injected endothelial cells integrate into the dorsal aorta and become subendothelial mesenchymal cells expressing SMA.43 Several groups have shown that Flk1+ (vascular endothelial growth factor receptor-2) cells derived from embryonic stem cells can differentiate into endothelial cells and subsequently into smooth muscle cells with loss of Flk1 expression.44–46 Smooth muscle cells derived from endothelial cells have been shown to incorporate into sites of neovascularization in the adult.46,47 Endothelial to smooth muscle cell transdifferentiation is not restricted to embryonic cells, because several groups have shown that adult endothelial cells can differentiate into smooth muscle cells.31,37,38
Other studies have observed that neointimal smooth muscle cells in atherosclerosis and restenosis seem to arise from endothelial cells.20,31,48 Notch receptors and Jagged1 are expressed in both endothelial and periendothelial cells, and a marked increase in expression of Jagged1 and Jagged2 and Notch2 through 4 has been described in the regenerating endothelium after balloon injury of the rat carotid artery.49 Mature vascular endothelium can give rise to smooth muscle cells via EMT using a mechanism that requires cell-cell contact.31 This is in line with our findings showing that Jagged1-Notch interactions on adjacent cells induce EMT. It is tempting to speculate that Notch activation may play a role in smooth muscle formation in neointimal growth or during vascular development.
In mammals, members of the TGF-ß superfamily as well as various extracellular matrix proteins seem to play an important role in EMT during endocardial cushion formation.18,50 However, both TGF-ß–dependent and TGF-ß–independent events are involved, and TGF-ß does not induce SMA in microvascular endothelial cells and umbilical vein endothelium.19,51 In our in vitro system, Notch-mediated EMT is restricted to cells expressing activated Notch, and TGF-ß1 and -ß2 do not seem to be essential for Notch-mediated transformation. However, additional investigation is required to verify whether other members of the TGF-ß superfamily are involved and whether EMT requires crosstalk between the Notch and TGF-ß pathways in vivo. Nevertheless, our data indicate a potential role for the Notch pathway in endocardial cushion development and possibly in vascular smooth muscle development and neointimal formation.
|
Acknowledgments |
|---|
This research was supported by grants to A.K. from the Heart and Stroke Foundation of British Columbia and the Yukon and to A.K. and P.H. from the National Cancer Institute of Canada with funds from the Canadian Cancer Society and the Terry Fox Foundation. M.N. was supported by a fellowship from Fondazione Italiana per la Ricerca sul Cancro, a fellowship from the Canadian Institutes of Health Research, and a Research Trainee Award from the Michael Smith Foundation for Health Research. G.M. was supported by a Research Trainee Award from the Michael Smith Foundation for Health Research. P.H. is a Scholar of the Canadian Institutes of Health Research and the Michael Smith Foundation for Health Research. A.K. is a Clinician-Scientist of the Canadian Institutes of Health Research and a Scholar of the Michael Smith Foundation for Health Research. We thank Denise McDougal for assistance with flow cytometry and cell sorting.
|
Footnotes |
|---|
*Both authors contributed equally to this study.
Original received November 3, 2003; revision received February 6, 2004; accepted February 16, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Bray S, Furriols M. Notch pathway: making sense of suppressor of hairless. Curr Biol. 2001; 11: R217–R221.[CrossRef][Medline] [Order article via Infotrieve]
2. Joutel A, Tournier-Lasserve E. Notch signalling pathway and human diseases. Semin Cell Dev Biol. 1998; 9: 619–625.[CrossRef][Medline] [Order article via Infotrieve]
3. Li L, Krantz ID, Deng Y, Genin A, Banta AB, Collins CC, Qi M, Trask BJ, Kuo WL, Cochran J, Costa T, Pierpont ME, Rand EB, Piccoli DA, Hood L, Spinner NB. Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1. Nat Genet. 1997; 16: 243–251.[CrossRef][Medline] [Order article via Infotrieve]
4. Oda T, Elkahloun AG, Pike BL, Okajima K, Krantz ID, Genin A, Piccoli DA, Meltzer PS, Spinner NB, Collins FS, Chandrasekharappa SC. Mutations in the human Jagged1 gene are responsible for Alagille syndrome. Nat Genet. 1997; 16: 235–242.[CrossRef][Medline] [Order article via Infotrieve]
5. McElhinney DB, Krantz ID, Bason L, Piccoli DA, Emerick KM, Spinner NB, Goldmuntz E. Analysis of cardiovascular phenotype and genotype-phenotype correlation in individuals with a JAG1 mutation and/or Alagille syndrome. Circulation. 2002; 106: 2567–2574.
6. Eldadah ZA, Hamosh A, Biery NJ, Montgomery RA, Duke M, Elkins R, Dietz HC. Familial tetralogy of Fallot caused by mutation in the jagged1 gene. Hum Mol Genet. 2001; 10: 163–169.
7. Krebs LT, Xue Y, Norton CR, Shutter JR, Maguire M, Sundberg JP, Gallahan D, Closson V, Kitajewski J, Callahan R, Smith GH, Stark KL, Gridley T. Notch signaling is essential for vascular morphogenesis in mice. Genes Dev. 2000; 14: 1343–1352.
8. Uyttendaele H, Ho J, Rossant J, Kitajewski J. Vascular patterning defects associated with expression of activated Notch4 in embryonic endothelium. Proc Natl Acad Sci U S A. 2001; 98: 5643–5648.
9. Leong KG, Hu X, Li L, Noseda M, Larrivee B, Hull C, Hood L, Wong F, Karsan A. Activated Notch4 inhibits angiogenesis: role of ß1-integrin activation. Mol Cell Biol. 2002; 22: 2830–2841.
10. Xue Y, Gao X, Lindsell CE, Norton CR, Chang B, Hicks C, Gendron-Maguire M, Rand EB, Weinmaster G, Gridley T. Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1. Hum Mol Genet. 1999; 8: 723–730.
11. McCright B, Lozier J, Gridley T. A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency. Development. 2002; 129: 1075–1082.[Medline] [Order article via Infotrieve]
12. Sakata Y, Kamei CN, Nakagami H, Bronson R, Liao JK, Chin MT. Ventricular septal defect and cardiomyopathy in mice lacking the transcription factor CHF1/Hey2. Proc Natl Acad Sci U S A. 2002; 99: 16197–16202.
13. Donovan J, Kordylewska A, Jan YN, Utset MF. Tetralogy of Fallot and other congenital heart defects in Hey2 mutant mice. Curr Biol. 2002; 12: 1605–1610.[CrossRef][Medline] [Order article via Infotrieve]
14. Iso T, Hamamori Y, Kedes L. Notch signaling in vascular development. Arterioscler Thromb Vasc Biol. 2003; 23: 543–553.
15. Loomes KM, Taichman DB, Glover CL, Williams PT, Markowitz JE, Piccoli DA, Baldwin HS, Oakey RJ. Characterization of Notch receptor expression in the developing mammalian heart and liver. Am J Med Genet. 2002; 112: 181–189.[CrossRef][Medline] [Order article via Infotrieve]
16. Loomes KM, Underkoffler LA, Morabito J, Gottlieb S, Piccoli DA, Spinner NB, Baldwin HS, Oakey RJ. The expression of Jagged1 in the developing mammalian heart correlates with cardiovascular disease in Alagille syndrome. Hum Mol Gen. 1999; 8: 2443–2449.
17. Icardo JM, Manasek FJ. Cardiogenesis: Development, Mechanisms and Embryology. 2nd ed. New York, NY: Raven Press; 1992.
18. Eisenberg LM, Markwald RR. Molecular regulation of atrioventricular valvuloseptal morphogenesis. Circ Res. 1995; 77: 1–6.
19. Paranya G, Vineberg S, Dvorin E, Kaushal S, Roth SJ, Rabkin E, Schoen FJ, Bischoff J. Aortic valve endothelial cells undergo transforming growth factor-ß–mediated and non-transforming growth factor-ß-mediated transdifferentiation in vitro. Am J Pathol. 2001; 159: 1335–1343.
20. Beranek JT. Vascular endothelium-derived cells containing smooth muscle actin are present in restenosis. Lab Invest. 1995; 72: 771.[Medline] [Order article via Infotrieve]
21. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science. 1999; 284: 770–776.
22. Weinmaster G, Roberts VJ, Lemke G. A homolog of Drosophila Notch expressed during mammalian development. Development. 1991; 113: 199–205.[Abstract]
23. Valsecchi C, Ghezzi C, Ballabio A, Rugarli EI. JAGGED2: a putative Notch ligand expressed in the apical ectodermal ridge and in sites of epithelial-mesenchymal interactions. Mech Dev. 1997; 69: 203–207.[CrossRef][Medline] [Order article via Infotrieve]
24. Karsan A, Yee E, Poirier GG, Zhou P, Craig R, Harlan JM. Fibroblast growth factor-2 inhibits endothelial cell apoptosis by Bcl-2-dependent and independent mechanisms. Am J Pathol. 1997; 151: 1775–1784.[Abstract]
25. Karsan A, Yee E, Harlan JM. Endothelial cell death induced by tumor necrosis factor- is inhibited by the Bcl-2 family member, A1. J Biol Chem. 1996; 271: 27201–27204.
26. Dorovini-Zis K, Prameya R, Bowman PD. Culture and characterization of microvascular endothelial cells derived from human brain. Lab Invest. 1991; 64: 425–436.[Medline] [Order article via Infotrieve]
27. Ear T, Giguere P, Fleury A, Stankova J, Payet MD, Dupuis G. High efficiency transient transfection of genes in human umbilical vein endothelial cells by electroporation. J Immunol Methods. 2001; 257: 41–49.[CrossRef][Medline] [Order article via Infotrieve]
28. Hsieh JJ, Henkel T, Salmon P, Robey E, Peterson MG, Hayward SD. Truncated mammalian Notch1 activates CBF1/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2. Mol Cell Biol. 1996; 16: 952–959.[Abstract]
29. Hoodless PA, Pye M, Chazaud C, Labbe E, Attisano L, Rossant J, Wrana JL. FoxH1 (Fast) functions to specify the anterior primitive streak in the mouse. Genes Dev. 2001; 15: 1257–1271.
30. Dejana E. Endothelial adherens junctions: implications in the control of vascular permeability and angiogenesis. J Clin Invest. 1997; 100: S7–S10.[Medline] [Order article via Infotrieve]
31. Frid MG, Kale VA, Stenmark KR. Mature vascular endothelium can give rise to smooth muscle cells via endothelial-mesenchymal transdifferentiation: in vitro analysis. Circ Res. 2002; 90: 1189–1196.
32. Nakajima Y, Mironov V, Yamagishi T, Nakamura H, Markwald RR. Expression of smooth muscle -actin in mesenchymal cells during formation of avian endocardial cushion tissue: a role for transforming growth factor ß3. Dev Dyn. 1997; 209: 296–309.[CrossRef][Medline]
[Order article via Infotrieve]
33. Icardo JM, Manasek FJ. An indirect immunofluorescence study of the distribution of fibronectin during the formation of the cushion tissue mesenchyme in the embryonic heart. Dev Biol. 1984; 101: 336–345.[CrossRef][Medline] [Order article via Infotrieve]
34. Ffrench-Constant C, Hynes RO. Patterns of fibronectin gene expression and splicing during cell migration in chicken embryos. Development. 1988; 104: 369–382.
35. Ataliotis P, Mercola M. Distribution and functions of platelet-derived growth factors and their receptors during embryogenesis. Int Rev Cytol. 1997; 172: 95–127.[Medline] [Order article via Infotrieve]
36. Hellstrom M, Kalen M, Lindahl P, Abramsson A, Betsholtz C. Role of PDGF-B and PDGFR-ß in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development. 1999; 126: 3047–3055.[Abstract]
37. Arciniegas E, Sutton AB, Allen TD, Schor AM. Transforming growth factor ß1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro. J Cell Sci. 1992; 103: 521–529.[Abstract]
38. Ishisaki A, Hayashi H, Li AJ, Imamura T. Human umbilical vein endothelium-derived cells retain potential to differentiate into smooth muscle-like cells. J Biol Chem. 2003; 278: 1303–1309.
39. Boyer AS, Ayerinskas II, Vincent EB, McKinney LA, Weeks DL, Runyan RB. TGFß2 and TGFß3 have separate and sequential activities during epithelial-mesenchymal cell transformation in the embryonic heart. Dev Biol. 1999; 208: 530–545.[CrossRef][Medline] [Order article via Infotrieve]
40. Camenisch TD, Molin DG, Person A, Runyan RB, Gittenberger-de Groot AC, McDonald JA, Klewer SE. Temporal and distinct TGFß ligand requirements during mouse and avian endocardial cushion morphogenesis. Dev Biol. 2002; 248: 170–181.[CrossRef][Medline] [Order article via Infotrieve]
41. Krantz ID, Smith R, Colliton RP, Tinkel H, Zackai EH, Piccoli DA, Goldmuntz E, Spinner NB. Jagged1 mutations in patients ascertained with isolated congenital heart defects. Am J Med Genet. 1999; 84: 56–60.[CrossRef][Medline] [Order article via Infotrieve]
42. Drake CJ, Hungerford JE, Little CD. Morphogenesis of the first blood vessels. Ann N Y Acad Sci. 1998; 857: 155–179.[CrossRef][Medline] [Order article via Infotrieve]
43. DeRuiter MC, Poelmann RE, VanMunsteren JC, Mironov V, Markwald RR, Gittenberger-de Groot AC. Embryonic endothelial cells transdifferentiate into mesenchymal cells expressing smooth muscle actins in vivo and in vitro. Circ Res. 1997; 80: 444–451.
44. Ema M, Faloon P, Zhang WJ, Hirashima M, Reid T, Stanford WL, Orkin S, Choi K, Rossant J. Combinatorial effects of Flk1 and Tal1 on vascular and hematopoietic development in the mouse. Genes Dev. 2003; 17: 380–393.
45. Yamashita J, Itoh H, Hirashima M, Ogawa M, Nishikawa S, Yurugi T, Naito M, Nakao K. Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature. 2000; 408: 92–96.[CrossRef][Medline] [Order article via Infotrieve]
46. Marchetti S, Gimond C, Iljin K, Bourcier C, Alitalo K, Pouyssegur J, Pages G. Endothelial cells genetically selected from differentiating mouse embryonic stem cells incorporate at sites of neovascularization in vivo. J Cell Sci. 2002; 115: 2075–2085.
47. Yurugi-Kobayashi T, Itoh H, Yamashita J, Yamahara K, Hirai H, Kobayashi T, Ogawa M, Nishikawa S, Nakao K. Effective contribution of transplanted vascular progenitor cells derived from embryonic stem cells to adult neovascularization in proper differentiation stage. Blood. 2003; 101: 2675–2678.
48. Majesky MW, Schwartz SM. An origin for smooth muscle cells from endothelium? Circ Res. 1997; 80: 601–603.[Medline] [Order article via Infotrieve]
49. Lindner V, Booth C, Prudovsky I, Small D, Maciag T, Liaw L. Members of the Jagged/Notch gene families are expressed in injured arteries and regulate cell phenotype via alterations in cell matrix and cell-cell interaction. Am J Pathol. 2001; 159: 875–883.
50. Nakajima Y, Yamagishi T, Hokari S, Nakamura H. Mechanisms involved in valvuloseptal endocardial cushion formation in early cardiogenesis: roles of transforming growth factor (TGF)-ß and bone morphogenetic protein (BMP). Anat Rec. 2000; 258: 119–127.[CrossRef][Medline] [Order article via Infotrieve]
51. Boyer AS, Erickson CP, Runyan RB. Epithelial-mesenchymal transformation in the embryonic heart is mediated through distinct pertussis toxin-sensitive and TGFß signal transduction mechanisms. Dev Dyn. 1999; 214: 81–91.[CrossRef][Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  H. Liu, S. Kennard, and B. Lilly NOTCH3 Expression Is Induced in Mural Cells Through an Autoregulatory Loop That Requires Endothelial-Expressed JAGGED1 Circ. Res., February 27, 2009; 104(4): 466 - 475. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Morrow, S. Guha, C. Sweeney, Y. Birney, T. Walshe, C. O'Brien, D. Walls, E. M. Redmond, and P. A. Cahill Notch and Vascular Smooth Muscle Cell Phenotype Circ. Res., December 5, 2008; 103(12): 1370 - 1382. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Quillard, S. Coupel, F. Coulon, J. Fitau, M. Chatelais, M.C. Cuturi, E. Chiffoleau, and B. Charreau Impaired Notch4 Activity Elicits Endothelial Cell Activation and Apoptosis: Implication for Transplant Arteriosclerosis Arterioscler. Thromb. Vasc. Biol., December 1, 2008; 28(12): 2258 - 2265. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Venkatesh, K.-S. Park, A. Harrington, L. Miceli-Libby, J. K. Yoon, and L. Liaw Cardiovascular and Hematopoietic Defects Associated With Notch1 Activation in Embryonic Tie2-Expressing Populations Circ. Res., August 15, 2008; 103(4): 423 - 431. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Niessen, Y. Fu, L. Chang, P. A. Hoodless, D. McFadden, and A. Karsan Slug is a direct Notch target required for initiation of cardiac cushion cellularization J. Cell Biol., July 28, 2008; 182(2): 315 - 325. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Jin, E. M. Hansson, S. Tikka, F. Lanner, C. Sahlgren, F. Farnebo, M. Baumann, H. Kalimo, and U. Lendahl Notch Signaling Regulates Platelet-Derived Growth Factor Receptor-{beta} Expression in Vascular Smooth Muscle Cells Circ. Res., June 20, 2008; 102(12): 1483 - 1491. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Niessen and A. Karsan Notch Signaling in Cardiac Development Circ. Res., May 23, 2008; 102(10): 1169 - 1181. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. T. Holderfield and C. C.W. Hughes Crosstalk Between Vascular Endothelial Growth Factor, Notch, and Transforming Growth Factor-{beta} in Vascular Morphogenesis Circ. Res., March 28, 2008; 102(6): 637 - 652. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Wu, Q. Zhou, L. Huang, A. Sun, K. Wang, Y. Zou, and J. Ge Ageing-exaggerated proliferation of vascular smooth muscle cells is related to attenuation of Jagged1 expression in endothelial cells Cardiovasc Res, March 1, 2008; 77(4): 800 - 808. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. G. Leong, K. Niessen, I. Kulic, A. Raouf, C. Eaves, I. Pollet, and A. Karsan Jagged1-mediated Notch activation induces epithelial-to-mesenchymal transition through Slug-induced repression of E-cadherin J. Exp. Med., November 26, 2007; 204(12): 2935 - 2948. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Liu, V. R. Joag, and A. I. Gotlieb The Emerging Role of Valve Interstitial Cell Phenotypes in Regulating Heart Valve Pathobiology Am. J. Pathol., November 1, 2007; 171(5): 1407 - 1418. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. T Butcher and R. R Markwald Valvulogenesis: the moving target Phil Trans R Soc B, August 29, 2007; 362(1484): 1489 - 1503. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Gridley Notch signaling in vascular development and physiology Development, August 1, 2007; 134(15): 2709 - 2718. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Niessen and A. Karsan Notch signaling in the developing cardiovascular system Am J Physiol Cell Physiol, July 1, 2007; 293(1): C1 - C11. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Arciniegas, M. G. Frid, I. S. Douglas, and K. R. Stenmark Perspectives on endothelial-to-mesenchymal transition: potential contribution to vascular remodeling in chronic pulmonary hypertension Am J Physiol Lung Cell Mol Physiol, July 1, 2007; 293(1): L1 - L8. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Bolos, J. Grego-Bessa, and J. L. de la Pompa Notch Signaling in Development and Cancer Endocr. Rev., May 1, 2007; 28(3): 339 - 363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Fischer, C. Steidl, T. U. Wagner, E. Lang, P. M. Jakob, P. Friedl, K.-P. Knobeloch, and M. Gessler Combined Loss of Hey1 and HeyL Causes Congenital Heart Defects Because of Impaired Epithelial to Mesenchymal Transition Circ. Res., March 30, 2007; 100(6): 856 - 863. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. M.S. van den Akker, D. G.M. Molin, P. P.W.M. Peters, S. Maas, L. J. Wisse, R. van Brempt, C. J. van Munsteren, M. M. Bartelings, R. E. Poelmann, P. Carmeliet, et al. Tetralogy of Fallot and Alterations in Vascular Endothelial Growth Factor-A Signaling and Notch Signaling in Mouse Embryos Solely Expressing the VEGF120 Isoform Circ. Res., March 30, 2007; 100(6): 842 - 849. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. L. Aranguren, A. Luttun, C. Clavel, C. Moreno, G. Abizanda, M. A. Barajas, B. Pelacho, M. Uriz, M. Arana, A. Echavarri, et al. In vitro and in vivo arterial differentiation of human multipotent adult progenitor cells Blood, March 15, 2007; 109(6): 2634 - 2642. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. C. Aird Phenotypic Heterogeneity of the Endothelium: II. Representative Vascular Beds Circ. Res., February 2, 2007; 100(2): 174 - 190. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. B. Rutenberg, A. Fischer, H. Jia, M. Gessler, T. P. Zhong, and M. Mercola Developmental patterning of the cardiac atrioventricular canal by Notch and Hairy-related transcription factors Development, November 1, 2006; 133(21): 4381 - 4390. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Doi, T. Iso, H. Sato, M. Yamazaki, H. Matsui, T. Tanaka, I. Manabe, M. Arai, R. Nagai, and M. Kurabayashi Jagged1-selective Notch Signaling Induces Smooth Muscle Differentiation via a RBP-J{kappa}-dependent Pathway J. Biol. Chem., September 29, 2006; 281(39): 28555 - 28564. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Noseda, Y. Fu, K. Niessen, F. Wong, L. Chang, G. McLean, and A. Karsan Smooth Muscle {alpha}-Actin Is a Direct Target of Notch/CSL Circ. Res., June 23, 2006; 98(12): 1468 - 1470. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Watanabe, H. Kokubo, S. Miyagawa-Tomita, M. Endo, K. Igarashi, K. i. Aisaki, J. Kanno, and Y. Saga Activation of Notch1 signaling in cardiogenic mesoderm induces abnormal heart morphogenesis in mouse Development, May 1, 2006; 133(9): 1625 - 1634. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Milan, A. C. Giokas, F. C. Serluca, R. T. Peterson, and C. A. MacRae Notch1b and neuregulin are required for specification of central cardiac conduction tissue Development, March 15, 2006; 133(6): 1125 - 1132. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. G. Leong and A. Karsan Recent insights into the role of Notch signaling in tumorigenesis Blood, March 15, 2006; 107(6): 2223 - 2233. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. G. Leblanc, J. F. Meschia, D. T. Stuss, and V. Hachinski Genetics of Vascular Cognitive Impairment: The Opportunity and the Challenges Stroke, January 1, 2006; 37(1): 248 - 255. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Noseda, K. Niessen, G. McLean, L. Chang, and A. Karsan Notch-Dependent Cell Cycle Arrest Is Associated With Downregulation of Minichromosome Maintenance Proteins Circ. Res., July 22, 2005; 97(2): 102 - 104. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. J. Briegel, H. S. Baldwin, J. A. Epstein, and A. L. Joyner Congenital heart disease reminiscent of partial trisomy 2p syndrome in mice transgenic for the transcription factor Lbh Development, July 15, 2005; 132(14): 3305 - 3316. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. R. Carlson, Y. Yan, X. Wu, M. T. Lam, G. L. Tang, L. J. Beverly, L. M. Messina, A. J. Capobianco, Z. Werb, and R. Wang Endothelial expression of constitutively active Notch4 elicits reversible arteriovenous malformations in adult mice PNAS, July 12, 2005; 102(28): 9884 - 9889. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Noseda, L. Chang, G. McLean, J. E. Grim, B. E. Clurman, L. L. Smith, and A. Karsan Notch Activation Induces Endothelial Cell Cycle Arrest and Participates in Contact Inhibition: Role of p21Cip1 Repression Mol. Cell. Biol., October 15, 2004; 24(20): 8813 - 8822. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. MacKenzie, P. Duriez, B. Larrivee, L. Chang, I. Pollet, F. Wong, C. Yip, and A. Karsan Notch4-induced inhibition of endothelial sprouting requires the ankyrin repeats and involves signaling through RBP-J{kappa} Blood, September 15, 2004; 104(6): 1760 - 1768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Liebner, A. Cattelino, R. Gallini, N. Rudini, M. Iurlaro, S. Piccolo, and E. Dejana {beta}-Catenin is required for endothelial-mesenchymal transformation during heart cushion development in the mouse J. Cell Biol., August 2, 2004; 166(3): 359 - 367. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||